Eukaryotic cells use approximately 5% of their genes to synthesize lipids. Such a heavy portion is invested because of the indispensable functions of lipids in cells. With their unique structures, lipids form bilayers to segregate the internal constituents from the extracellular environment as well as to compartmentalize discrete organelles. In addition to their barrier function, lipids are also used for energy storage in lipid droplets and as messengers in signal transduction and molecular recognition processes.
Cardiolipin is a diphosphatidylglycerol lipid exclusively found in the mitochondrial inner membrane. CL regulates enzymatic activities involved in electron transport and oxidative phosphorylation. This unique lipid consists of four unsaturated acyl chains and a polar head with two negative charges, having the structure:

Interaction of CL with the protein cytochrome c (cyt c) activates the peroxidase activity of the protein and triggers mitochondria-mediated apoptosis. During apoptosis, the distribution of CL changes, which consequently affects ATP synthesis in mitochondria. Meanwhile, the level of CL decreases during apoptosis in a time-dependent manner, correlating with the release of cyt c to the cytosol (intracellular fluid or cytoplasmic matrix found inside cells) and the generation of reactive oxygen species.
In addition to having an important role in the apoptosis pathway, the CL level of mitochondria is also of clinical significance. The depletion of CL is a critical indicator of aging and Barth syndrome, as well as a number of diseases associated with mitochondrial respiratory function including heart ischemia, reperfusion, gliomas, cardiac hypertrophy, and cardiac failure. Tangier disease is caused by the abnormal enhanced production of CL. Parkinson's disease, HIV-1, and various cancers are reported to be associated with the abnormalities of CL. Therefore, developing effective methods for detection and quantification of CL is of high importance.
Prior art examples of various methods for quantifying cardiolipin include those disclosed by William Kenneth Lang (US 2004/0096903 A1), Wonhwa Cho (US 2012/0225447 A1), Ruey-min Lee (US 2006/0172958 A1), Fatih M. Uckun (US 2001/0044442 A1), and Robert E. Davis (US 2001/0021526 A1). However, these prior art examples generally face several problems, such as lacking a standard protocol, involving extra substrates, and involving sophisticated methods.
Particularly, specific detection of CL among numerous phospholipids is not trivial. Lipidomics profiling by high-resolution liquid chromatograph mass spectrometry (LC-MS) has recently been developed for quantitative analysis of CL. This powerful method requires sophisticated instrumentation and experienced operators, which limit the scope of its application.
Optical detection by fluorescence, on the other hand, is a relatively simple and accessible method while providing superior sensitivity. In the early 1980s, a fluorescent dye, 10-N-nonyl acridine orange (NAO) was introduced for CL detection and mitochondria staining, NAO having the structure:

The green fluorescence of NAO is decreased in the presence of CL. However, the quantification of CL by NAO is not realistic, as both the excitation and emission maxima are dependent on the dye concentration, and the linear relationship can be established only when the NAO/CL molar ratio is equal to 2. To quantify mitochondria with NAO, tortuous steps are involved, including mitochondria fixation, long time incubation, and centrifugation. Furthermore, NAO suffers from small Stokes shift and poor water-solubility, making NAO less appealing for use in biological systems. The working mechanism of NAO is still unclear and the performance is difficult to improve, even through different approaches. Although there are numerous drawbacks, NAO has been used for many years, even without a standard protocol, because no alternative has been developed so far.